Force-frequency Response Research Articles

Development of an in vitro platform for the analysis of contractile and calcium dynamics in single human myotubes.

In vitro myotube cultures are widely used as models for studying muscle pathophysiology, but their limited maturation and heterogeneity pose significant challenges for functional analyses. While they remain the gold standard for studying muscle function in vitro, myotube cultures do not fully recapitulate the complexity and native features of muscle fibers, which may compromise their ability to predict in vivo outcomes. To promote maturation and decrease heterogeneity, we have incorporated engineered structures into myotube cultures, based on a PDMS thin layer with micrometer-sized grooves (μGrooves) placed over a glass substrate. Different sizes and shapes of μGrooves were tested for their ability to promote alignment and fusion of myoblasts and enhance their differentiation into myotubes. A 24 hour electrical field stimulation protocol (4 V, 6 ms, 0.1 Hz) was used to further promote myotube maturation, after which several myotube features were assessed, including myotube alignment, width, fusion index, contractile function, and calcium handling. Our results indicate superior calcium and contractile performance in μGrooved myotubes, particularly with the 100 μm-width 700 μm-long geometry (7 : 1). This platform generated homogeneous and isolated myotubes that reproduced native muscle features, such as excitation-contraction coupling and force-frequency responses. Overall, our 2D muscle platform enables robust high-content assays of calcium dynamics and contractile readouts with increased sensitivity and reproducibility compared to traditional myotube cultures, making it particularly suitable for screening therapeutic candidates for different muscle pathologies.

The RyR1 P3528S Substitution Alters Mouse Skeletal Muscle Contractile Properties and RyR1 Ion Channel Gating.

The recessive Ryanodine Receptor Type 1 (RyR1) P3527S mutation causes mild muscle weakness in patients and increased resting cytoplasmic [Ca2+] in transformed lymphoblastoid cells. In the present study, we explored the cellular/molecular effects of this mutation in a mouse model of the mutation (RyR1 P3528S). The results were obtained from 73 wild type (WT/WT), 82 heterozygous (WT/MUT) and 66 homozygous (MUT/MUT) mice with different numbers of observations in individual data sets depending on the experimental protocol. The results showed that WT/MUT and MUT/MUT mouse strength was less than that of WT/WT mice, but there was no difference between genotypes in appearance, weight, mobility or longevity. The force frequency response of extensor digitorum longus (EDL) and soleus (SOL) muscles from WT/MUT and MUT/MUT mice was shifter to higher frequencies. The specific force of EDL muscles was reduced and Ca2+ activation of skinned fibres shifted to a lower [Ca2+], with an increase in type I fibres in EDL muscles and in mixed type I/II fibres in SOL muscles. The relative activity of RyR1 channels exposed to 1 µM cytoplasmic Ca2+ was greater in WT/MUT and MUT/MUT mice than in WT/WT mice. We suggest the altered RyR1 activity due to the P2328S substitution could increase resting [Ca2+] in muscle fibres, leading to changes in fibre type and contractile properties.

Effects of high-intensity interval training (HIIT) on skeletal muscle atrophy, function, and myokine profile in diabetic myopathy

PurposeDiabetes is a metabolic disorder characterized by chronic hyperglycemia due to insulin deficiency and/or loss of its action. Diabetic myopathy causes functional limitations in diabetic patients. The beneficial effects of high-intensity interval training (HIIT) are widely reported. We have hypothesized that HIIT application would prevent the development of diabetic myopathy. MethodsMale, Wistar albino rats (10 W) were randomly divided into four groups (1)Control(C), (2)Diabetes(DM), (3)Training(HIIT), and (4)Diabetes + Training(DM + HIIT). Streptozotocin(60 mg/kg) was injected for the induction of diabetes. The maximum exercise capacity(MEC) of animals was determined by an incremental load test. HIIT protocol (4 min 85–95 % MEC, 2 min 40–50 % MEC, 6 cycles, 5 days/week) was applied for 8 weeks. In the end, functional parameters, atrophy, and resistance to fatigue in soleus and EDL muscles were evaluated. IL-6, FNDC5, and myonectin levels were measured in EDL, soleus, and serum. ResultsWe observed atrophy, fatigue sensitivity, and proinflammatory alterations (IL-6 increase) in the EDL samples due to diabetic myopathy which were not observed in the soleus samples. HIIT application prevented the aforementioned detrimental alterations. Both force-frequency response and parallelly the twitch amplitude increased significantly in the DM + HIIT group. Half relaxation time (DT50) increased in both exercising and sedentary diabetics. FNDC5 was significantly higher in the exercising animals in soleus samples. Myonectin was significantly higher in the soleus muscle only in the DM + HIIT group. ConclusionCurrent findings show that diabetic myopathy develops earlier in glycolytic-fast-twitch fibers(EDL) than in oxidative-slow-twitch fibers(soleus). Furthermore, HIIT application prevents atrophy in skeletal muscle, increases resistance to fatigue, and has an anti-inflammatory effect. New findingsThe current study analyzes the myokine profile and skeletal muscle function under the effect of diabetes HIIT-type exercise. We also measured maximal exercise capacity and tailored the exercise program individually according to the result. Diabetic myopathy is an important complication of diabetes yet still, it is not understood completely.Our results show that HIIT-type training would be beneficial in diabetic myopathy but further investigation is needed to understand the whole molecular mechanism.

A novel dynamic model on nonlinear vibrations of functionally graded graphene platelet reinforced rotating pretwisted composite blade considering subsonic airflow excitation and blade-casing rubbing

A novel dynamic model of the functionally graded graphene platelet (FGGP) reinforced rotating pretwisted composite blade under the aerodynamic force and blade-casing rubbing is established for the first time. The blade is simplified to a FGGP reinforced rotating pretwisted composite cantilever plate. The dynamic model of the axial excitation produced by considering the aerodynamic force in the tip clearance includes two trigonometric functions with different frequencies. The subsonic airflow is considered as a transverse excitation which is derived by utilizing the vortex lattice method (VLM). The dynamic model expresses the changes of the non-uniform axial and contact forces through the tip clearance and rotating pretwisted plate-casing rubbing when the shaft is eccentric. Based on Rayleigh-Ritz method, the linear frequencies and mode shapes are obtained for the FGGP reinforced rotating pretwisted plate. The influences of the graphene distribution pattern, rotating pretwisted plate-casing rubbing, axial excitation in the tip clearance and geometric parameter on the frequencies are investigated. Using the obtained mode shapes, von-Karman type nonlinear geometric assumptions and Lagrange equation, the differential governing equations of motion are derived for the FGGP reinforced rotating pretwisted plate. The nonlinear vibrations under the 1:1 internal resonance at two critical rotating speeds is studied by using Runge-Kutta method. The amplitude-frequency and force-frequency response curves under the low and high critical rotating speeds are investigated by using numerical calculations. The obtained results demonstrate the influence of the rotating speed, frequency ratios and incoming flow speeds on the nonlinear vibrations of the FGGP reinforced rotating pretwisted plate. The dynamic model of the rubbing-impact for the rotating blade provides a theoretical basis for the blade-casing rubbing analysis. At the same time, this study is also used as a theoretical guidance to reduce the damage of the blade-casing rubbing and blade design.

Prediction of Surface Location Error Considering the Varying Dynamics of Thin-Walled Parts during Five-Axis Flank Milling

Surface location error (SLE) caused by forced vibration is a key factor to determine the quality of the finished part. When machining thin-walled structures with sculptured surfaces, the complicated milling process is significantly influenced by the vibration due to the flexibility of the part. The dynamics of the part are dominant and vary with the material removal during machining. This paper presents a prediction method of SLE considering the varying dynamics of thin-walled parts in five-axis flank milling. The in-process part is decomposed into unmachined and machined portions, which are both modelled based on the thin-plate theory. The dynamics models of the two portions are coupled using the substructure method. Coordinate transformation based on the screw theory and the general cutting dynamics model for five-axis flank milling is employed to transform the cutting force vectors and frequency response function (FRF) to the same coordinate system for the prediction of SLE. The proposed method is validated with five-axis flank milling tests and SLE measurements on a thin-walled twisted part. It is shown that the average error of the proposed method for SLE prediction is less than 5 μm, and the calculation is almost 8 times faster than the typical finite element method.

Nonlinear dynamics analysis of a dual-rotor-bearing-casing system based on a modified HB-AFT method

This paper presents a modified harmonic balance-alternating frequency/time domain (HB-AFT) method to analyze the nonlinear dynamics of a dual-rotor-bearing-casing system. The motion equations of a dual-rotor-bearing-casing system subjected to the unbalanced excitations of the two rotors are formulated with 284 degrees of freedom, in which, the complex nonlinear factors of the inter-shaft bearing such as exponential nonlinear restoring force, radial clearance and varying stiffness excitation are taken into consideration. The HB-AFT method is modified by applying the AFT procedure to obtain the harmonic expending coefficients of the harmonic balance residuals rather than that of the nonlinear terms, hence, the Jacobian matrix is calculated programmatically and efficiently, as a result the performance of the HB-AFT method is improved greatly. The primary resonance of the system is analyzed by employing the three-dimensional diagrams of amplitude-frequency responses of each node of the system, including the inner casing and the outer casing. In addition, the amplitude-frequency response curves with separated frequencies and orbits of the two rotors are also discussed. Furthermore, the pseudo-arclength continuation procedure is introduced into the modified HB method, then all periodic solution branches of the system including the unstable solutions are obtained. The results show that there are two resonant peaks in the amplitude-frequency response curves of the system and the vibration of each part of the system, i.e., LPC, LPT, HPC, HPT, inner casing and outer casing, is synchronous. Besides, the nonlinear dynamic behaviors such as vibration jumping, bi-stable and resonance hysteresis are revealed. Accordingly, the dynamic load of the inter-shaft bearing is calculated. The results show that the evolution of the inter-shaft bearing’s dynamic load is associated with the vibration responses of the two rotors. The nonlinear characteristics of the force-frequency responses of the inter-shaft bearing are consistent with that of the two rotors and casings. In addition, with the increase of the inter-shaft bearing clearance, the nonlinear characteristics of the force-frequency responses of the inter-shaft bearing are more significant. In comparison with the Newmark method, the modified HB-AFT method proposed in this paper has a great advantage in computing efficiency, and it can also grasp all solution branches of the system including the unstable solutions. Consequently, it has great potential to deal with high-dimensional systems with complex nonlinearities and multifrequency excitations in practical engineering.

The Unsteady-State Response of Tires to Slip Angle and Vertical Load Variations

The tire is the only part that connects the vehicle and the road surface. Many important properties of vehicles are related to the mechanical properties of tires, such as handling stability, braking safety, vertical vibration characteristics, and so on. Although a great deal of research on tire dynamics has been completed, mainly focusing on steady-state tire force and moment characteristics, as well as linear unsteady force characteristics, less research has been conducted on nonlinear unsteady characteristics, especially when the vertical load changes dynamically. Therefore, the main purpose of this paper is to improve the tire unsteady-state model and verify it by experiment. To achieve this goal, we first study the nonlinear unsteady tire cornering theoretical model and obtain clear force and torque frequency response functions. Then, based on the results of the theoretical model, a high-precision and high-efficiency semi-physical model is developed. Finally, model identification and accuracy verification are carried out based on the bench test data. The model developed in this paper has high accuracy, and it significantly improves the expression of the aligning torque, which helps to improve the virtual simulation of transient conditions, such as vehicle handling and dynamic load conditions.

Troponin C mutations alter twitch kinetics and force-frequency relationship of fast and slow twitch skeletal muscle

Troponin C (TnC) is critically involved in the contraction of striated muscle. The role of TnC on the mechanical process of skeletal muscle has been thoroughly investigated. However, the impact of TnC mutations on the force-frequency relationship (FFR) of skeletal muscle is yet to be investigated. The objective of this study is to determine the effect of TnC mutations on the force-frequency relationship and contractile kinetics in fast and slow-twitch skeletal muscle. To investigate this key question, we measured the extensor digitorum longus (EDL) and soleus force-frequency response in a murine model with a TnC mutation (expressed after TnCL48QAAV or TnCD73NAAV injection) that causes increased calcium sensitivity (L48Q) or a decreased calcium sensitivity (D73N) and compared the FFR to the wildtype counterparts.

Nonlinear vibrations of rotating pretwisted composite blade reinforced by functionally graded graphene platelets under combined aerodynamic load and airflow in tip clearance

The primary resonance and nonlinear vibrations of the functionally graded graphene platelet (FGGP)-reinforced rotating pretwisted composite blade under combined the external and multiple parametric excitations are investigated with three different distribution patterns. The FGGP-reinforced rotating pretwisted composite blade is simplified to the rotating pretwisted composite cantilever plate reinforced by the functionally graded graphene platelet. It is novel to simplify the leakage of the airflow in the tip clearance to the non-uniform axial excitation. The rotating speed of the steady state adding a small periodic perturbation is considered. The aerodynamic load subjecting to the surface of the plate is simulated as the transverse excitation. Utilizing the first-order shear deformation theory, von Karman nonlinear geometric relationship, Lagrange equation and mode functions satisfying the boundary conditions, three-degree-of-freedom nonlinear ordinary differential equations of motion are derived for the FGGP-reinforced rotating pretwisted composite cantilever plate under combined the external and multiple parametric excitations. The primary resonance and nonlinear dynamic behaviors of the FGGP-reinforced rotating pretwisted composite cantilever plate are analyzed by Runge–Kutta method. The amplitude–frequency response curves, force–frequency response curves, bifurcation diagrams, maximum Lyapunov exponent, phase portraits, waveforms and Poincare map are obtained to investigate the nonlinear dynamic responses of the FGGP-reinforced rotating pretwisted composite cantilever plate under combined the external and multiple parametric excitations.

Development of a force actuator for hybrid model tests using super-twisting sliding mode control

Hybrid model testing is a solution to overcome the limitations of small-scale model testing, such as scaling conflict or infrastructure limits, still retaining the real physical phenomena’ complexity. In hybrid model testing, the physical and numerical substructures interact with each other through a network of sensors and actuators. So, to apply the desired effects in the physical substructure, precise control of the actuators is necessary. In this paper, a robust control technique will be employed (super-twisting sliding mode control) to design the force actuator controller for hybrid model testing of vessels in a wave tank. First, the force actuator system dynamics is detailed, consisting of a servomotor controlling the force in a line attached to the vessel model. The methodology and design for the super-twisting sliding mode controller are described, taking into account the uncertain friction torque, the vessel motion and the system delays. Finally, the proposed actuator’s performance is validated experimentally in a simpler environment: The servomotor is mounted on a linear guideway to simulate the vessel’s horizontal displacement in waves. We analyzed the force actuator step response and frequency response (0.1–2.0 Hz) with disturbance motions varying from 0.5 to 1.5 Hz. The developed actuator showed good results (mean absolute error inferior to 0.1 N in all tests), where the high-frequency error spikes (up to 0.33 N) should not have a significant effect on the hybrid model tests’ slow dynamics. We conclude that the force actuator will be a valuable tool for hybrid model testing.

Efficient joint identification and fluted segment modelling of shrink-fit tool assemblies by updating extended tool models

In milling, the dynamic behavior of the tool center point is crucial for estimating surface quality of the workpiece as well as the process stability behavior. Experimental-analytical receptance coupling can be used for predicting the tool tip dynamics but requires accurate analytical modelling of the holder-tool assembly. This includes the reliable identification of the holder-tool joint properties as well as the correct modelling of the fluted segment of end mills. However, the modelling effort associated with accurately representing the dynamic behavior of the fluted segment is significant. In addition, the joint identification requires a reference tool tip frequency response function of the tool assembly clamped in the machine spindle. This is inefficient and can also lead to incorrect estimation of joint properties. This paper provides an efficient method for joint identification and fluted section modelling using an offline, free–free excitation approach. The objective of this paper is to enable a direct comparison of the dynamic behavior of the freely constrained analytical tool assembly model with that of the real freely constrained tool assembly. The comparison of displacement to force frequency response at certain points on the tool assembly allows for the identification of tool model parameters such as the joint properties and effective diameter of the fluted segment. The comparability is realized by extending the analytical holder-tool beam model to include the receptance model of the standard spindle-holder interface. In this study, as an example, a thermal shrink-fit holder-tool beam model is extended to include an HSK-A63 interface. Subsequently, frequency response functions at two points on the real freely constrained tool assembly are measured in order to identify the joint stiffness and effective diameter of the fluted segment using the corresponding proposed formulations. The updated holder-tool model is then coupled with a 4-axis milling machine and validated. Despite the reduced modelling effort, a good prediction accuracy could be achieved for different holder-tool combinations.

Design and Validation of a Two-Degree-of-Freedom Powered Ankle-Foot Orthosis with Two Pneumatic Artificial Muscles

Powered ankle-foot orthosis (PAFO) is a field of wearable robotics that improves the lives of people of old age or with physical impairments by aiding in the wearer's ankle joint movements. Most of the PAFOs developed thus far offer only one degree-of-freedom (dof), which uses the talocrural joint alone as the axis of rotation, where the emphasis is on moving forward. However, because this type of wearable robotics has evolved, developing PAFOs for functional rehabilitation has become necessary. This enhances the quality of walking rather than providing simple rehabilitation. The subtalar joint is responsible for the rotation of the inversion and eversion of the ankle, enabling balanced walking in humans, and is an important part of balance training for elderly people or stroke patient rehabilitation. Therefore, we developed a 2-dof PAFO that uses a calculated spatial formula of the subtalar joint based on anatomical data. In recognition of the fact that the pneumatic artificial muscle (PAM) can be used within the contracting range alone because of the instinct of the PAM, we analyzed the workspace of the fabricated PAFO through kinematic analysis and verified the possibility of using the PAFO during the gait cycle. Experiments were also conducted on the closed-loop force frequency response using a sliding mode control of the solenoid valve to validate the control characteristics. Lastly, clinical experiments with healthy subjects were conducted for validation in wearing conditions.

The effect of temperature acclimation on the force-frequency relationship and adrenergic sensitivity of the ventricle of two populations of juvenile sockeye salmon.

We tested the hypothesis that cardiorespiratory differences known to exist among adult sockeye salmon populations also exist in the juveniles. To test this hypothesis, we compared cardiac contractility and adrenergic responsiveness of juvenile sockeye salmon from two geographically isolated populations that were reared from eggs under common garden conditions and at two acclimation temperatures (5°C and 14°C). However, we found no substantive differences in the force-frequency response (FFR) and the cardiac pumping capacity of juveniles from Weaver Creek and Chilko River populations, even when we considered wild-reared juveniles from one of the populations. An unexpected discovery for all fish groups at 5°C was a rather flat FFR during tonic β-adrenergic stimulation (βAR) stimulation. Curiously, while active tension nearly doubled with maximum βAR stimulation at low pacing frequencies for all fish groups, a negative FFR with maximum βAR stimulation meant that this inotropic benefit was lost at the highest pacing frequency (0.8Hz). Active tension with tonic βAR stimulation was similar at 14°C, but maximum pacing frequency doubled and all fish groups displayed a modest negative FFR. Maximum βAR stimulation again doubled active tension and this benefit was retained even at the highest pacing frequency (1.6Hz) at 14°C. Even though subtle population differences were apparent for the FFR and pumping capacity, their biological significance is unclear. What is clear, however, is that the cardiac pumping capacity of juvenile sockeye would benefit more from βAR stimulation swimming at 15°C than when swimming at 5°C.

Force transmissibility and frequency response of a flexible shaft–disk rotor supported by a nonlinear suspension system

In this work, the nonlinear dynamic and force transmissibility characteristics of a flexible shaft–disk rotor system supported by suspension system with nonlinear stiffness and damping are investigated numerically. The distributed variables representing the elastic motions of the shaft are discretized using the Assumed Mode Method (AMM) and the equations of motion (EOM) are subsequently obtained using Lagrange’s equation. The Harmonic Balance (HB) method combined with a continuation scheme is used to determine the nonlinear frequency response and force transmissibility curves due to disk unbalance force. The effect of the bearing stiffness nonlinearity on the system dynamics when supported by stiff and soft linear bearing stiffness is investigated. In addition, the influence of the bearing damping nonlinearity is investigated and compared with that due to linear damping.

The Role of Thin Filament Calcium Sensitivity in Modulating Relaxation Time of Soleus and EDL Skeletal Muscle

The mechanical process of skeletal muscle relaxation; myofilament detachment, calcium sequestration, and Ca2+-Troponin dissociation, has been well understood for some time. However, many questions about the physiological and pathophysiological role of relaxation time (RT) remain. The objective of this study is to determine if relaxation time is a dynamic property that can be modulated by altering the calcium sensitivity of the thin filament.Our data suggests that the relaxation time of Het mice (utrn+/-,MDX, a moderate Duchenne Muscular Dystrophy (DMD) phenotype) is significantly increased in soleus muscle (predominantly slow-twitch) across a broad frequency range and also at some higher frequencies in EDL muscle (predominantly fast-twitch) compared to WT, age matched mice. We hypothesize that relaxation is a dynamic and controllable process and delayed relaxation is an effect of DMD disease progression.The present research plan was designed to study the effects of altered relaxation time on wildtype and Het mouse soleus and EDL contractile properties using an adeno-associated virus to express engineered troponin C with increased or decreased calcium sensitivities. Contractile properties are assessed by twitch force, tetanic force, time to peak, half relaxation time, and comparing force-frequency response curves. While the basic mechanical process of skeletal muscle relaxation is known, our data demonstrates a need for further investigation into the physiological control of this process and the role that thin filament calcium sensitivity (Troponin C) plays in the regulation of relaxation.

Nonlinear Structural Control Analysis of an Offshore Wind Turbine Tower System

This paper investigates the vibration control, stability, and energy transfer of the offshore wind turbine tower system with control force and nonlinearity terms. A nonlinear proportional derivative (NPD) controller was connected to the system to reduce a high oscillation amplitude and to transfer the energy in the wind turbine system. Furthermore, the averaging method and Poincaré maps were used with respect to the controlled system to study the stability and bifurcation analysis in the worst resonance cases. The curves of force response and frequency response were plotted before and after the control unit was added to the wind turbine system. In addition, we discuss the performances of the control parameters on the vibration magnitudes. Numerical simulations were carried out with Maple and Matlab algorithms to confirm the analytical results. The results show the effectiveness of the NPD controller in suppressing the nonlinear oscillations of the wind turbine system.

Engineered Cardiac Tissues Generated in the Biowire II: A Platform for Human-Based Drug Discovery.

Recent advances in techniques to differentiate human induced pluripotent stem cells (hiPSCs) hold the promise of an unlimited supply of human derived cardiac cells from both healthy and disease populations. That promise has been tempered by the observation that hiPSC-derived cardiomyocytes (hiPSC-CMs) typically retain a fetal-like phenotype, raising concern about the translatability of the in vitro data obtained to drug safety, discovery, and development studies. The Biowire II platform was used to generate 3D engineered cardiac tissues (ECTs) from hiPSC-CMs and cardiac fibroblasts. Long term electrical stimulation was employed to obtain ECTs that possess a phenotype like that of adult human myocardium including a lack of spontaneous beating, the presence of a positive force-frequency response from 1 to 4 Hz and prominent postrest potentiation. Pharmacology studies were performed in the ECTs to confirm the presence and functionality of pathways that modulate cardiac contractility in humans. Canonical responses were observed for compounds that act via the β-adrenergic/cAMP-mediated pathway, eg, isoproterenol and milrinone; the L-type calcium channel, eg, FPL64176 and nifedipine; and indirectly effect intracellular Ca2+ concentrations, eg, digoxin. Expected positive inotropic responses were observed for compounds that modulate proteins of the cardiac sarcomere, eg, omecamtiv mecarbil and levosimendan. ECTs generated in the Biowire II platform display adult-like properties and have canonical responses to cardiotherapeutic and cardiotoxic agents that affect contractility in humans via a variety of mechanisms. These data demonstrate that this human-based model can be used to assess the effects of novel compounds on contractility early in the drug discovery and development process.

Abstract 479: Cardiomyopathy Phenotypes Observed in Human Engineered Heart Tissue Depend on Functional Maturation

Human iPSC-based models of cardiomyopathies have become increasingly prevalent. These models enable the study of genotype-phenotype relationships within a human context and have already led to new insights. A potential limitation of iPSC-derived cardiomyocytes (iPSC-CMs) is their varying degrees of functional maturation with respect to electrophysiology, calcium handling, and contractility. Variable levels of maturity may explain how different groups investigating hypertrophic cardiomyopathy (HCM) mutations in iPSC cardiomyocytes have arrived at conflicting results. Nevertheless, whether or not advanced functional maturation truly impacts disease phenotype in iPSC cardiomyocyte models remains an open question. Using a novel protocol that combines pacing and physiological media in a three-dimensional engineered heart tissue (EHT) derived from decellularized myocardium, we are able to mature human engineered heart tissues within three weeks to recapitulate key functional characteristics of adult heart tissue, such as a robustly positive force-frequency response, a strong post-rest potentiation, potent calcium handling and excitation-contraction handling, and mature isometric twitches that closely match that of intact adult human ventricular myocardium. Using our platform, we investigated the effect of maturation on the functional consequence of a classic variant (E62Q) in the thin filament protein alpha-tropomyosin (TPM1). Initial characterization of TPM1 E62Q in EHTs show a hypercontractile phenotype compared to its isogenic wild-type line, with limited changes in calcium handling. Preliminary data on maturing TPM1 E62Q EHTs show that additional hallmarks of disease may develop with maturation such as arrhythmogenicity, hypertrophy, and fibrosis. In our work, we develop a method for advanced maturation of hiPSC-CMs in EHTs and show the dependence on maturation status of progressive hallmarks of HCM in human EHTs compared to EHTs grown under conditions standard for the field.

Hemodynamic Stimulation Using the Biomimetic Cardiac Tissue Model (BCTM) Enhances Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell (hiPSC)-derived cardio­myocytes (hiPSC-CMs) hold great promise for cardiovascular disease modeling and regenerative medicine. However, these cells are both structurally and functionally ­immature, primarily due to their differentiation into cardiomyocytes occurring under static culture which only reproduces biomolecular cues and ignores the dynamic hemo­dynamic cues that shape early and late heart development during cardiogenesis. To evaluate the effects of hemodynamic stimuli on hiPSC-CM maturation, we used the biomimetic cardiac tissue model to reproduce the hemodynamics and pressure/volume changes associated with heart development. Following 7 days of gradually increasing stimulation, we show that hemodynamic loading results in (a) enhanced alignment of the cells and extracellular matrix, (b) significant increases in genes associated with physiological hypertrophy, (c) noticeable changes in sarcomeric organization and potential changes to cellular metabolism, and (d) a significant increase in fractional shortening, suggestive of a positive force frequency response. These findings suggest that culture of hiPSC-CMs under conditions that accurately reproduce hemodynamic cues results in structural orga­nization and molecular signaling consistent with organ growth and functional maturation.

The role of RyR2 oxidation in the blunted frequency-dependent facilitation of Ca2+ transient amplitude in rabbit failing myocytes.

Defective Ca2+ regulation plays a key role in the blunted force-frequency response in heart failure (HF). Since HF is commonly associated with oxidative stress, we studied whether oxidation of ryanodine receptor (RyR2) contributes to this defect. In control ventricular myocytes, oxidative stress induced formation of disulfide bonds between RyR2 subunits: intersubunit cross-linking (XL). Western blot analysis and Ca2+ imaging revealed a strong positive correlation between RyR2 XL and sarcoplasmic reticulum (SR) Ca2+ leak. These results illustrate that RyR2 XL can be used as a sensitive indicator of RyR2 dysfunction during oxidative stress. HF myocytes were in a state of oxidative stress since they exhibited an increase in reactive oxygen species (ROS) level, a decrease in ROS defense and an overall protein oxidation. These myocytes were also characterized by RyR2 XL and increased SR Ca2+ leak. Moreover, the frequency-dependent increase of Ca2+ transient amplitude was suppressed due to the inability of the SR to maintain Ca2+ load at high pacing rates. Because SR Ca2+ load is determined by the balance between SR Ca2+ uptake and leak, the blunted frequency-dependent inotropy in HF can be mediated by ROS-induced SR Ca2+ leak. Preventing RyR2 XL in HF myocytes decreased SR Ca2+ leak and increased Ca2+ transients at high pacing rate. We also studied whether RyR2 oxidation alone can cause the blunted frequency-dependent facilitation of Ca2+ transient amplitude in control myocytes. When RyR2 XL was induced in control myocytes to a similar level seen in HF, an increase of Ca2+ transient amplitude at high pacing rate was significantly suppressed. These results suggest that SR Ca2+ leak induced by RyR2 oxidation can play an important role in the blunted frequency-dependent inotropy of HF.